Takahashi et al., Int Arch Allergy Immunol 2001, 124, 454-460, discloses a study of the effects of mutations in the active site and the N-glycosylation site of a recombinant pro-form of the house dust mite allergen Der f 1 (rproDer f1) on secretion and protease activity. A mutant of rproDer f 1 having a mutation in the N-glycosylation site, N133Q (the numbering is consistently based on the pro-form of the protein), and a mutant additionally having a mutation in the active site, C115S/N133Q, were studied. C115 and N133 in proDer f 1 corresponds to C114 and N132 in proDer p 1. The study showed that N-glycosylation is essential for secretion in insect SF9 cells but not in Pichia pastoris, and that disulfide bonds are essential for secretion in Pichia pastoris. Also, the study showed that the pro-sequence could be removed in vitro by incubation under acidic conditions, and that the mature C115S/N133Q mutant had a low protease activity. Indirect evidence was obtained to support the disulfide bond formation between Cys4 and Cys8.
Furthermore, a contemporary study has shown that the protease activity and reactivity to IgE of the wild type and the N133Q mutant were restored after autocatalytic removal of the pro-sequences by the in vitro method (Yasuhara et al., Clinical and experimental Allergy, 2001, Volume 31, pages 116-124.
Topham et al., Protein Engineering, Vol. 7, No. 7, pp 869-894, 1994 discloses a computer model of the three-dimensional structure of mature house dust mite allergen Der p 1 modelled from its amino acid sequence and its homology to three known structures of papain, actinidin and papaya proteinase Ω, all from the cysteine protease family. Various computer modelling programs were used.
Draborg et al., Scandinavian Journal of Immunology 59, 623 (Abstract), 2004, discloses expression and secretion of a recombinant form of proDer p 1 in Saccharomyces cerevisiae by fusion with the pro-enzyme with a fungal signal peptide. The N-glycosylation site of Der p 1 was mutated. The non-glycosylated recombinant proDer p 1 molecule revealed immunological similarity to the native Der p 1 molecule. A three-dimensional structure of Der p 1 was modelled in silico and used to predict epitopes.